A broad goal of the proposed research is to gain new information about the molecular organization and functions of eukaryotic plasma membranes using a novel physical fractionation approach. Previous work established the feasibility of using cell freeze-fracture as a tool to separate the plasma membrane bilayer into split membrane fractions. This monolayer splitting approach was recently improved, the new method being called "double labeled membrane splitting" (DBLAMS). In DBLAMS spectroscopic assays of split preparations are used to quantify the fractions of extracellular and cytoplasmic membrane "halves." In contrast to existing chemical and physical approaches, the fortes of DBLAMS are that it is uniquely a physical fractionation technique, it is a quantitative method, and it can be used with cell surfaces of irregular and unknown morphology. Experiments outlined in this proposal will establish the limits of DBLAMS in its future application to biomembrane studies in general, while answering residual, intractable questions about the structure of the human erythrocyte (RBC) membrane. Assays for membrane splitting have been devised where native hemoglobin serves as a marker for the cytoplasmic side of the membrane and a fluoresceinated concanavalin A as a marker for the extracellular side. A primary objective of this proposal is to apply DBLAMS and a newly developed single membrane splitting method to studies of the transmembrane distributions of lipids and polypeptides, both native and probe molecules, in intact RBCs and lysed RBC ghost preparations. Details of lipid distributions obtained by DBLAMS will provide insight into the mechanisms of membrane permeability for neutral lipids and minimally hydrophilic molecules such as steroid hormones, certain pharmaceuticals, and cholesterol. Details of polypeptide distributions will answer questions about the transbilayer organization of transport, recognition, and signaling sites in the membrane and provide a basis for understanding the mechanisms of their functions. The new sensitive and rapid gel staining procedure for membrane glycoproteins outlined in this proposal should facilitate these studies. Because DBLAMS can evaluate cells of irregular morphology, a condition associated with many RBC pathologies, the examination of diseased cells should also provide new and useful information.